This application is a National Stage Application filed under 35 U.S.C.xc2xa7371 of PCT/GB00/02364, filed on Jun. 16, 2000.
This invention relates to newly identified spermine:peptide-based surfactant compounds, to the use of such compounds and to processes for their preparation. The invention also relates to the use of the spermine:peptide-based surfactant compounds to facilitate the transfer of compounds into cells for drug delivery.
Surfactants are substances that markedly affect the surface properties of a liquid, even at low concentrations. For example surfactants will significantly reduce surface tension when dissolved in water or aqueous solutions and will reduce interfacial tension between two liquids or a liquid and a solid. This property of surfactant molecules has been widely exploited in industry, particularly in the detergent and oil industries. In the 1970s a new class of surfactant molecule was reported, characterised by two hydrophobic chains with polar heads which are linked by a hydrophobic bridge (Deinega, Y et al., Kolloidn. Zh. 36, 649, 1974). These molecules, which have been termed xe2x80x9cgeminixe2x80x9d (Menger, F M and Littau, C A, J.Am.Chem.Soc. 113, 1451, 1991), have very desirable properties over their monomeric equivalents. For example they are highly effective in reducing interfacial tension between oil and water based liquids and have a very low critical micelle concentration.
Cationic surfactants have been used inter alia for the transfection of polynucleotides into cells in culture, and there are examples of such agents available commercially to scientists involved in genetic technologies (for example the reagent Tfx(trademark)-50 for the transfection of eukaryotic cells available from Promega Corp. WI, USA).
The efficient delivery of DNA to cells in vivo, either for gene therapy or for antisense therapy, has been a major goal for some years. Much attention has concentrated on the use of viruses as delivery vehicles, for example adenoviruses for epithelial cells in the respiratory tract with a view to corrective gene therapy for cystic fibrosis (CF). However, despite some evidence of successful gene transfer in CF patients, the adenovirus route remains problematic due to inflammatory side-effects and limited transient expression of the transferred gene. Several alternative methods for in vivo gene delivery have been investigated, including studies using cationic surfactants. Gao,X et al. (1995) Gene Ther. 2, 710-722 demonstrated the feasibility of this approach with a normal human gene for CF transmembrane conductance regulator (CFTR) into the respiratory epithelium of CF mice using amine carrying cationic lipids. This group followed up with a liposomal CF gene therapy trial which, although only partially successful, demonstrated the potential for this approach in humans (Caplen, N J. et al., Nature Medicine, 1, 39-46, 1995). More recently other groups have investigated the potential of other cationic lipids for gene delivery, for example cholesterol derivatives (Oudrhiri,N et al. Proc.Natl.Acad.Sci. 94, 1651-1656, 1997). This limited study demonstrated the ability of these cholesterol based compounds to facilitate the transfer of genes into epithelial cells both in vitro and in vivo, thereby lending support to the validity of this general approach.
These studies, and others, show that in this new field of research there is a continuing need to develop novel low-toxicity surfactant molecules to facilitate the effective transfer of polynucleotides into cells both in vitro for transfection in cell-based experimentation and in vivo for gene therapy and antisense treatments. The present invention seeks to overcome the difficulties exhibited by existing compounds.
The invention relates to spermine:peptide-based surfactant compounds having a spermine backbone and having the general structure of formula (I): 
where R1 and R3 are hydrogen and R2 and R4, which may be the same or different, are peptide groups formed from one or more amino acids linked together, in a linear or branched manner, by amide (CONH) bonds and further linked to the spermine backbone by amide bonds, having the general formula (II): 
where p1 is 0 to 5 and p2 is 1 to 5, preferably 1; and the values for p3 and p4, which may be the same or different, are from 0 to 5, preferably 0;
A1, A3 and A4, which may be the same or different, are amino acids selected from serine, lysine, ornithine, threonine, histidine, cysteine, arginine and tyrosine; and
A2 is an amino acid selected from lysine, ornithine and histidine;
and R5 and R6 are saturated or unsaturated hydrocarbyl groups having up to 24 carbon atoms and linked to the spermine backbone by an amide or an amine (NCH2) linkage;
or
where R1 and R3 are hydrogen, R2 and R4, which may be the same or different are saturated or unsaturated hydrocarbyl groups having up to 24 carbon atoms and linked to the spermine backbone by amide or amine bonds, and R5 and R6, which may be the same or different, are peptide groups of formula (II) linked to the spermine backbone by amide bonds;
or
a salt, preferably a pharmaceutically acceptable salt thereof.
When used herein, the term xe2x80x9chydrocarbylxe2x80x9d refers to a group having from 1 to 24 carbon atoms which may be in a straight chain or a branched chain and include a saturated carbocyclic ring having from 3 to 6 carbon atoms and which chain may contain unsaturation (double and/or triple carbon-carbon bonds).
The amide linkages between the amino acids A1, A2 and A3 in the peptide group of formula (II) are standard peptide bonds (xcex1 bonds), unless the amino acid is a diamine, for example lysine or ornithine, where the linkage may involve either of the two amine groups. For example, where A1 is lysine, the linkage to the amino acid A2 may be a standard alpha amide bond, or an epsilon (xcex5) amide bond involving the amine of the lysine side chain. Similarly where A1 is ornithine the amide bond linking A1 to A2 may be an alpha bond or a delta (xcex4) bond that is created using the amine on the side chain of the ornithine amino acid residue.
Preferably, the compound is symmetrical, that is R1 and R3 are the same, R2 and R4 are the same, and R5 and R6 are the same. Symmetrical spermine:peptide-based surfactant compounds of the invention are xe2x80x9cgeminixe2x80x9d surfactants.
In a preferred embodiment A1 in the group of formula (II) is serine or threonine, prefereably serine. Preferably A3 and A4 in the group of formula (II) are lysine, ornithine, histidine or arginine.
In a further preferred embodiment the hydrocarbyl group is selected from:
xe2x80x94CO(CH2)10CH3 
xe2x80x94CO(CH2)12CH3 
xe2x80x94CO(CH2)14CH3 
xe2x80x94CO(CH2)16CH3 
xe2x80x94CO(CH2)18CH3 
xe2x80x94CO(CH2)22CH3 
xe2x80x94CO(CH2)7CHxe2x95x90CH(CH2)5CH3 
xe2x80x94CO(CH2)7CHxe2x95x90CH(CH2)7CH3 
xe2x80x94CO(CH2)7CHxe2x95x90CHCH2CHxe2x95x90CH (CH2)4CH3 
xe2x80x94CO(CH2)7(CHxe2x95x90CHCH2)3CH3 
xe2x80x94CO(CH2)3CHxe2x95x90CH(CH2CHxe2x95x90CH)3(CH2)4CH3 
xe2x80x94CO(CH2)7CHxe2x95x90CH(CH2)5CH3 Trans
xe2x80x94CO(CH2)7CHxe2x95x90CH(CH2)7CH3 Trans
xe2x80x94CO(CH2)8CHCH3(CH2)7CH3 
xe2x80x94COCHOH(CH2)21CH3 
In another preferred embodiment the hydrocarbyl group is selected from:
xe2x80x94(CH2)11CH3 
xe2x80x94(CH2)13CH3 
xe2x80x94(CH2)15CH3 
xe2x80x94(CH2)17CH3 
xe2x80x94(CH2)19CH3 
xe2x80x94(CH2)23CH3 
xe2x80x94(CH2)8CHxe2x95x90CH (CH2)5CH3 
xe2x80x94(CH2)8CHxe2x95x90CH (CH2)7CH3 
xe2x80x94(CH2)8CHxe2x95x90CHCH2CHxe2x95x90CH (CH2)4CH3 
xe2x80x94(CH2)8(CHxe2x95x90CHCH2)3CH3 
xe2x80x94(CH2)4CHxe2x95x90CH(CH2CHxe2x95x90CH)3(CH2)4CH3 
xe2x80x94(CH2)8CHxe2x95x90CH(CH2)5CH3 Trans
xe2x80x94(CH2)8CHxe2x95x90CH(CH2)7CH3 Trans
xe2x80x94(CH2)9CHCH3(CH2)7CH3 
Compounds of the present invention may be prepared from readily available starting materials using synthetic peptide chemistry well known to the skilled person. The scheme shown in FIGS. 1a and 1b shows a general process for the synthesis of the compounds of the invention wherein the hydrocarbyl groups are linked to the spermine moiety by amine bonds and the scheme shown in FIGS. 2a and 2b shows a general process for the synthesis of the compounds of the invention wherein the carbonyl groups are linked to the spermine moiety by amide bonds.
The processes shown in FIGS. 1 and 2 are for the synthesis of symmetrical, that is xe2x80x9cgeminixe2x80x9d, spermine:peptide-based surfactants. Non-symmetrical spermine:peptide-based surfactants of the invention can be prepared by introducing asymmetry, for example at the primary amines of spermine, by using different protecting groups. Suitable nitrogen protecting groups are well known in the art and are described in, for example, xe2x80x9cProtective Groups in Organic Chemistryxe2x80x9d (T. W. Greene, Wiley-Interscience, New York, 2nd Edition, 1991).
Another aspect of the invention relates to methods for using the spermine:peptide-based surfactant compounds. Such uses include facilitating the transfer of DNA or RNA polynucleotides, or analogs thereof, into a eukaryotic or prokaryotic cell in vivo or in vitro. These uses include facilitating transfection of polynucleotides to achieve an antisense knock-out effect, for gene therapy and genetic immunization (for the generation of antibodies) in whole organisms. Other uses include employing the compounds of the invention to facilitate the transfection of polynucleotides into cells in culture when such transfer is required, in, for example, gene expression studies and antisense control experiments among others. The polynucleotides can be mixed with the compounds, added to the cells and incubated to allow polynucleotide uptake. After further incubation the cells can be assayed for the phenotypic trait afforded by the transfected DNA, or the levels of mRNA expressed from said DNA can be determined by Northern blotting or by using PCR-based quantitation methods for example the Taqmar(copyright) method (Perkin Elmer, Connecticut, USA). Compounds of the invention offer a significant improvement, typically between 3 and 6 fold, in the efficiency of cellular uptake of DNA in cells in culture, compared with compounds in the previous art. In the transfection protocol, the gemini compound may be used in combination with one or more supplements to increase the efficiency of transfection. Such supplements may be selected from, for example:
(i) a neutral carrier, for example dioleyl phosphatidylethanolamine (DOPE) (Farhood, H., et al (1985) Biochim. Biophys. Acta 1235 289);
(ii) a complexing reagent, for example the commercially available PLUS reagent (Invitrogen, Maryland, USA) or peptides, such as polylysine or polyornithine peptides or peptides comprising primarily, but not exclusively, basic amino acids such as lysine, ornithine and/or arginine. The list above is not intended to be exhaustive and other supplements that increase the efficiency of transfection are taken to fall within the scope of the invention.
In still another aspect, the invention relates to the transfer of genetic material in gene therapy using the compounds of the invention.
Yet another aspect of the invention relates to methods to effect the delivery of non-nucleotide based drug compounds into cells in vitro and in vivo using the compounds of the invention.
In a further aspect, the invention relates to methods to facilitate the transfer of a polynucleotide or an anti-infective compounds into prokaryotic or eukaryotic organism for use in anti-infective therapy.
The following definitions are provided to facilitate understanding of certain terms used frequently herein.
xe2x80x9cAmino acidxe2x80x9d refers to dipolar ions (zwitterions) of the form +H3NCH(R)CO2xe2x88x92. They are differentiated by the nature of the group R, and when R is different from hydrogen can also be asymmetric, forming D and L families. Amino acids may be natural or un-natural amino acids. There are 20 naturally occurring amino acids where the R group can be, for example, non-polar (e.g. alanine, leucine, phenylalanine) or polar (e.g. glutamic acid, histidine, arginine and lysine). In the case of un-natural amino acids R can be any other group which is not found in the amino acids found in nature.
xe2x80x9cPolynucleotidexe2x80x9d generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA. xe2x80x9cPolynucleotidesxe2x80x9d include, without limitation single-and double-stranded DNA, DNA that is a mixture of single-and double-stranded regions, single-and double-stranded RNA, and RNA that is mixture of single-and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions. In addition, xe2x80x9cpolynucleotidexe2x80x9d refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA. The term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons. xe2x80x9cModifiedxe2x80x9d bases include, for example, tritylated bases and unusual bases such as inosine. A variety of modifications have been made to DNA and RNA; thus, xe2x80x9cpolynucleotidexe2x80x9d embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells. xe2x80x9cPolynucleotidexe2x80x9d also embraces relatively short polynucleotides, often referred to as oligonucleotides.
xe2x80x9cTransfectionxe2x80x9d refers to the introduction of polynucleotides into cells in culture using methods involving the modification of the cell membrane either by chemical or physical means. Such methods are described in, for example, Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989). The polynucleotides may be linear or circular, single-stranded or double-stranded and may include elements controlling replication of the polynucleotide or expression of homologous or heterologous genes which may comprise part of the polynucleotide.
The invention will now be described by way of the following examples.